Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

Abstract

Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-␤-ManNAc3NAmA6Thr-1,4-␤-GlcNAc3NAcA-1,3-␤-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-␣-L-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the ⌬mmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The ⌬mmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins of Methanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway. While N-linked glycosylation was initially identified as an exclusively eukaryotic process, it is now well established that this pathway is present in the prokaryotic domains of Archaea and Bacteria as well. The posttranslational modification of proteins with N-linked glycans is believed to be much more widespread in Archaea than in Bacteria. In N-linked glycosylation systems, an oligosaccharyltransferase is required for the transfer of assembled oligosaccharides from the lipid carrier onto target proteins; genes encoding this key signature protein of the pathway have been identified in 166 out of 168 sequ...

Identification of a gene involved in the biosynthesis pathway of the terminal sugar of the archaellin N-linked tetrasaccharide in Methanococcus maripaludis

“…To test if the in-frame deletion of mmp1090, mmp1091 or mmp1092 resulted in a detectable truncation in the archaellin N-glycan, whole cell lysates of each mutant were first subjected to Western blot analysis, using anti-FlaB2 antibodies, as it has been shown previously that even small truncations of the N-glycan results in a faster migration of FlaB2 that is detectable on Western blots (Ding et al 2013;Jones et al 2012;Siu et al 2015;Vandyke et al 2009). As shown in Fig.…”

“…The knowledge of the biosynthesis of this dialdose is very limited and only one protein, AglV, the enzyme responsible for the transfer of the methyl group onto C5 via O-linkage, has been genetically identified in its biosynthesis pathway (Ding et al 2013). This is in contrast to the second and third sugars of the glycan where the biosynthesis pathways are now well known (Ding et al 2013;Jones et al 2012;Siu et al 2015). The involvement of MMP1090 as a UDP-glucose 4-epimerase in the biosynthesis pathway of the terminal sugar provides evidence that this unique sugar is probably synthesised from UDP-glucose or UDP-GlcNAc.…”

“…in DaglA and DaglL mutants, but not with monosaccharidemodified or non-glycosylated archaellins, i.e. DaglO or DaglB mutants (Siu et al 2015;Vandyke et al 2009). While archaella are assembled with a disaccharide or trisaccharide attached to the archaellins, motility is reduced in these mutants compared to that of wild type (WT) cells as measured in swarm plate assays.…”

“…The second region is located from mmp0350 to mmp0359. Several of the genes located in this area are involved in the biosynthesis of the second and third sugars of the glycan, including agl17 (mmp0350), agl18 (mmp0351), agl19 (mmp0352), agl20 (mmp0353) and agl21 (mmp0357) (Namboori and Graham 2008a;Siu et al 2015;VanDyke et al 2008). The gene for the OST, aglB (mmp1424) is found at a separate locus ).…”

Exploring Hydrogenotrophic Methanogenesis: a Genome Scale Metabolic Reconstruction of Methanococcus maripaludis

“…maripaludis utilizes a number of unusual coenzymes (methanofuran, H 4 MPT, coenzyme F 420 , coenzyme B, coenzyme M, and coenzyme F 430 ) as carbon and electron carriers during methanogenesis (60). It also contains recently characterized pathways for synthesizing a tetrasaccharide for N-linked glycosylation of archaellin (archaeal flagellin) (61) and multiple forms of archaeol, an archaeal membrane ether lipid (62). None of these pathways were included in our draft reconstruction, and few were completely present in the Model SEED database (63); thus, the bulk of these reactions were added manually.…”